Download lecture1

INSTRUMENTAL METHODS OF ANALYSIS (CHM 303)
FLUOROMETRY
Molecular Fluorescence
When a molecule absorbs electromagnetic energy, the energy is usually lost as heat by collision
process for certain molecules, (5-10%) especially when absorbing high energy radiation like UV,
only part of the energy is lost by collision; the electron then drops back to ground state by
emitting a photon of lower energy (longer wavelength) than the one absorbed.
The emitted radiation is usually in the visible region and at right angles to the incident
radiation; only visible rarely when absorbed radiation is of visible high energy (short
wavelength) in U.V region is the emitted radiation in U.V region. Molecules at room
temperature are in ground electronic state. It absorbs energy and goes to excited electronic
state. The groups of lines represent vibrational state. The entire process takes place in a very
short time (≈ 10-12 – 10-9 s). The absorption requires about 10-15 s while the collision takes about
10-8 s.
Excited state
hν
Energy
hν1
hν2
Ground state
The molecules emit energy hν by collision and drops to the lowest vibrational level of the
excited state. The probability of return to ground state with emission of photon is greatest at
this point. The electron thus emits a photon of energy hν2 at a longer wavelength which is lower
than hν1 absorbed. This emitted light is “fluorescence”. In some cases, the electron crosses over
to a triplet state (i.e. it becomes unpaired) before emitting a photon. This takes a longer time
>10-9 s. the emitted radiation is then called phosphorescence. Both processes are called
luminescence.
Relative fluorescent intensity
a
b
wavelength (λ)
a – excitation
b – fluorescent
Generally, Beer’s law applies to power of radiation transmitted by a substance or solution but
modification becomes necessary for fluorescence since radiation is emitted and not transmitted
Beer’s law:
ln T = ln P/Po = - abc
A = ln 1/T = ln Po/P = abc.
Fluorescence
F = k (Po - P) ……………………………….. (1)
Where F is the intensity of fluorescent radiation, k is constant (quantum yield), Po is incident
radiation and P is transmitted radiation.
I.e.
F ∞ (Po - P) i.e. radiation is absorbed.
From Beer’s law,
P = Po e-abc
Where Po is the power of incident radiation and P is the power of transmitted radiation.
Po – P = Po – Po e-abc ……………………………… (2)
= Po (1 – e-abc)……………………………… (3)
F = K (Po - P) = KPo (1 – e-abc)…………………. (4)
From (1), if no light is transmitted, i.e. all incident radiation is absorbed,
Fo = KPo ……………………………………………… (5)
From (4), F = Fo – Fo e-abc ……………………………………………… (6)
Fo – F/ Fo = e-abc
ln Fo – F/ Fo = -abc
ln Fo/ Fo – F = abc ………………………………. (7)
K is the fraction of incident radiation that is absorbed and depends on factors such as the
dimensions of the light beam, area of the solution irradiated, transmission band of filter before
photocell, spectral response of photocell etc. “a” is constant – absorptivity.
Hg vapour
1o filter
sample container
Lamp
Collimating lens
2o filter
Photocell
Galvanometer
“b” is the path length (thickness) of cell, “c” is concentration. When abc is small and negligible
compared to 1, (≤ 0.01), equation (4) becomes
F = 2.303 KPo . abc
= K’C
……………………… (8)
………………………………………
(Proved)
(9)
i.e. F ∞ C if abc ≤ 0.01. K’ is constant for a particular substance in a given instrument.
Equation (8) holds only for dilute solutions (when most of the radiation is transmitted, 92%) and
breaks down at higher concentration. As usual, there should be no dissociation or association of
molecules.
Chemical Structure and Fluorescence
In principle, any molecule that absorbs UV light radiation should fluoresce but not all do. The
greater the absorption by a molecule, the greater its fluorescence intensity. Many aromatic and
hetero compounds especially if they contain certain substituted groups. Compounds with
multiple conjugated double bonds are more favourable to fluoresce. Presence of one or more
electron donating group enhances fluorescence e.g. –OH, -NH2, -OCH3 etc. Polycyclic
compounds like vitamin K, purines and nucleosides and conjugated polyenes like vitamin A are
fluorescent. Groups like –NO2, -COOH, -CH2COOH, -Br, -I, and azo groups tend to inhibit
fluorescence. The nature of other substituent may affect the degree of fluorescence. The
fluorescence of many molecules is pH dependent as only the ionized or un-ionized form may be
fluorescent.
A compound that is not fluorescent may be converted to a fluorescent derivative e.g. nonfluorescent steroids may be converted to fluorescent compounds by dehydrating with conc.
H2SO4 which convert these cyclic alcohols to phenols. Similarly, dibasic acids e.g. maleic acid can
be reacted with β-naphtha in conc. H2SO4 to form a fluorescent derivative.
White et al developed fluorometric methods for many metals by forming chelates with organic
ligands. Antibodies can be made fluorescent by condensing them with fluorescein isocyanate
which reacts with free amino groups of the proteins.
Fluorescence Quenching
Some substances quench fluoresce. These substances compete for electronic excitation energy
and decrease the quantum yield (i.e. decrease the rate of conversion of absorbed energy to
fluorescent radiation). I- is a very effective quencher. I- and Br- substituent groups decrease
quantum yield. These substances may even be determined by measuring the extent of
fluorescence quenching. Some molecules do not fluoresce, whose bond dissociation energy is
less than that of incident radiation. Instead of getting excited, a bond is broken. Also, coloured
species in solution with fluorescing species may interfere by absorbing the fluorescent
radiation. This is “inner filter” effect. For example, in Na2CO3 solution, K2Cr2O7 has absorption
peaks at 245 and 348 nm, which overlap with excitation (275 nm) and emission (350 nm) peaks
of tryptophan and interferes.
Limitations
This arises because fluorometry is an extremely sensitive technique and can detect at ppb level
and the method is in fact limited to trace levels (a few ppm). Problems include instability of
dilute solutions due to adsorption onto container surface which leads to significant errors. The
problem is negligible in more concentrated solutions. Organic substances at < 1 ppm in organic
solvents are adsorbed onto glass surfaces. Addition of small amount of more polar solvent may
decrease it.
Quantitative Procedure
A series of standard solutions are prepared with slightly different concentration. The
fluorescent intensity (power) is measured and a calibration curve is plotted. The intensity of the
sample solution is also measured and concentration is read from calibration curve.
Infrared Spectrophotometry
The infrared region extends from a wavelength of 780nm to 1500nm (1.5µm) for near I.R and
1.5µm to 300µm for the far I.R, but the most useful region is from 2.5µm to 25µm which is
most frequently used for analysis.
Not all molecules can absorb in I.R region but only those with a change in dipole moment
(polarity) of the molecule. A diatomic molecule must have a permanent dipole although bigger
molecules do not. For example, NΞ N, H-H, O=O, without dipoles cannot absorb in I.R region. C
Ξ O has dipole moment and will absorb CO2 is symmetrical and has no net dipole and not
expected to absorb in I.R but by vibration, it develops dipole and absorbs. In the vibration mode
(a), there exist symmetry and no absorbance while in mode (b), there exist no symmetry and it
absorbs.
O <= C => O
O <=C <= O
(a)
(b)
I.R. Spectra and Molecular Structure
Vibrating groups absorbing in the I.R. region do so within certain wavelength region but the
exact wavelength depends on neighbouring groups. The peaks are sharper than in UV or visible
regions and easier to identify. Each molecule has a complete absorption spectrum unique to it
which is equivalent to a fingerprint of the molecule. Hence, the molecule can be identified.
Vibrational Transitions
Two types of molecular vibrations occur; stretching and bending, e.g. CO2.
O=C=O
I
O=C=O
II
O=C=O
III
I and II are stretching while III is bending. I will not lead to IR absorption while II and III will.
Bending may involve movement of a group of atoms within a molecule relative to the rest of
the molecule. Different types of bending occur: twisting, rocking, wagging, scissoring e.t.c.
IR absorption due to II and III of CO2 occur at “fundamental frequencies”. These are frequencies
at which intense absorption bands occur for complex molecules (ν1, ν2 etc). Less intense bands
called “overtones” may occur at multiples of the fundamental frequencies, e.g. (2ν1, 2ν2 etc).
There could also be combination tones at frequencies corresponding to sums of fundamental
frequencies e.g. (ν1 + ν2) etc.
Structure of Frequency
Vibrational frequency of the bond between given atoms or groups of atoms is characteristic and
not affected by molecular environment. It is therefore possible to obtain structural or
functional group information from IR spectra e.g. carbonyl group =C=O in alkanals and
alkanones absorbs at wave number 1700 cm-1 or wavelength 5.9µm. Structural changes can
cause minor shifts and changes in the absorption bands, which can provide additional
information. Correlation is possible between absorption frequency and types of bonds or
chemical groups.
There are four general regions in the IR spectrum;
1.
2.
Hydrogen stretching region (2.7 – 4.0 µm = λ). This includes stretching bands of O-H, NH, C-H and S-H bands.
Triple bond stretching region (λ = 4.5 – 5.0 µm). This includes stretching bands of C Ξ C
and C ΞN. Cumulated double bonds (C = C = C) also absorb in this region.
3.
Double bond stretching region (λ = 5.4 – 6.4 µm). This include C = C, C = O, C = N with C =
O at 5.9 µm.
Acids (-C=O-OH) and esters (-C=O-OR) absorb at lower wavelength while amides (-C=O-NH2)
absorb at longer wavelength with two peaks, hence discrimination is possible. These three
regions are the “functional group” region. The fourth region is the “fingerprint” region. This
is the single bond stretching and bending region. This includes not only C-H and N-H
stretching and bending but also vibrations of single bonds that connect groups such as
methyl (-CH3), (-CH2) and amine (-NH2) groups. Absorption in this region is very much
dependent on molecular environment and is unique for each molecule and leads to
identification of the molecule.
IR is mainly used for qualitative analysis i.e. for identification and structural analysis. It can
also be used for quantitative analysis of complex mixtures of similar compounds because
some absorption peaks for each compound occur at definite and selective wavelength and
have intensities directly proportional to the concentration of absorbing species.
Cells
Most common cells are cells of NaCl windows. The solvent used must not attack the
windows of the cell. NaCl cells must be protected from moisture and are stored in
desiccators. They require periodic polishing to remove “fogging” due to moisture
condensation.
AgCl windows are used for wet samples or aqueous solutions. These are soft and must be
protected from light as they slowly darken in visible light.
When samples therefore exist as pure liquid, they are run without dilution. This allows for
identification or confirmation of an unknown or new compound. The cell length must be
short (0.01 – 0.05 mm) to keep the absorbance within the optimum region. If solution of
sample is to be prepared, a fairly high concentration is required because no solvent is
completely transparent in the IR region and this keeps solvent absorbance at a minimum.
So, short path length ≤ 0.1mm.
Solids may be run as suspension or thick slurry in a viscous liquid having approximately
same refractive index to reduce light scattering. The sample is ground in the liquid, usually
Nujol (a mineral oil). If Nujol masks any C-H band, then chlorofluorocarbon greases are
used. This method is only for qualitative work since the slurry is difficult to reproduce for
quantitative work. Alternatively, the sample may be ground with KBr (transparent in IR
region) and pressed into a pellet for mounting.
For gases, a long path length (≈10cm) is required.
ATOMIC SPECTROMETRY
Emission
The principle depends on measurement of emitted radiant energy from sample solution
aspirated into the flame.
The instrument consists of the nebulizer, the burner, the optical system, detector, amplifier,
scale or recorder.
Nebulizer: This breaks the solution into a fine spray and introduces the spray into the flame
at a stable and reproducible rate. It must not be attacked by corrosive solutions and must
be easily cleaned. It is usually made of plastic.
Burner: It must provide a steady flame.
Optical System: This collects light from the steadiest part of the flame, renders it
monochromatic and focuses it onto the surface of a photosensitive detector. It consists of a
concave mirror placed behind the flame with its centre of curvature in the flame thereby
almost doubling the intensity of emitted radiation. For simple routine use, absorption filters
used for elements with simple spectrum and in absence of background emission from flame
transmits wide band as it cannot absorb radiation close to analytical line. Interference filters
are better but monochromators containing quartz prism or diffraction grating and narrow
slit-width, with very sensitive detecting circuits and amplifiers are best.